Serum from patients infected with hepatitis B virus (HBV) commonly have three distinct structures that contain the hepatitis B surface antigen (HBsAg): Dane particles, spherical particles, and filamentous particles. Dane particles are spheres that are 42 nm in diameter with a core that is 28 nm in diameter. The spherical particles have a diameter of about 22 nm. Filamentous particles have a diameter of about 22 nm and a length from about 50 nm to about 230 nm.
The particles contain three glycoprotein designated the major, middle, and large proteins. The hepatitis B surface antigen open reading frame of HBV-DNA is divided into three regions, pre-S 1, pre-S2, and S. This open reading frame encodes the major, middle, and large proteins. The complete amino acid sequence for the major protein is given in Valenzuela et al., Nature, 280:815-819 (1979). The amino acid sequence in Valenzuela et al. is 226 amino acids long and the amino acid positions referred to in this application refer to the amino acid sequence disclosed in Valenzuela et al. HBsAg contains several antigenic determinant,;, the most important of these are the a determinant, the d/y determinant, and the w/r determinant.
Hepatitis B vaccines have been used (extensively in humans in recent years. A typical vaccine, e.g., RECOMBIVAX HB vaccine, available from Merck, contains HBsAg of the adw subtype that has been produced recombinantly in yeast. As the human population becomes vaccinated, the virus is put under pressure to evolve around the vaccine. Mutants that evolve in response to the vaccine are termed "escape mutants." Current vaccines may not be effective against these (escape mutants. Also, some current immunodiagnostic tests may not detect these escape mutants.
Certain HBV escape mutants have been reported previously. A mutation at position 145 from glycine to arginine was reported by Carman et al., Lancet, 336:325-329 (1990). Also, a mutation where additional amino acids were inserted after amino acid 122 was reported in WO 95/21189. Other mutants are described in McMahon et al., Hepatology, 15(5):757-766 (1992). In addition, studies have been conducted on artificial mutant proteins. An example of this type of study is Mazngold et al., Virology, 211:535-543 (1995). Mangolet et al. made various mutations at positions 121, 124, 137, 139, 147, and 149.
The invention provides isolated mutant hepatitis B surface antigen proteins, fragments thereof, and particles containing these proteins. These mutant proteins, fragments, and particles can be used in improved vaccines and these mutant proteins, fragments, and particles and specific binding agents to them can be used in improved immunoassays.